Cell death by apoptosis is recognized as a major component of ischemia/reperfusion (I/R) injury. Activation of cell death pathways during I/R leads to loss of terminally differentiated cardiac myocytes, thus contributing to the development of heart failure. The Bcl-2 family proteins play an important role in regulating the mitochondrial pathway of apoptosis in the myocardium. BnipS is a pro-apoptotic member of the Bcl-2 family and is localized primarily to the mitochondria in myocardial cells. Overexpression of BnipS leads to mitochondrial dysfunction and cell death in various cell types, including neonatal cardiac myocytes. Elevated levels of BnipS protein have been reported in vivo in animal models of acute ischemia and heart failure. We have found that BnipS is expressed at substantially in the adult myocardium and our preliminary data indicate that BnipS plays a significant role in l/R-mediated cell death by activation of the mitochondrial pathway. Moreover, we have found that overexpression of BnipS causes extensive fragmentation of the mitochondrial network along with upregulation of autophagy, and that BnipS is subjected to proteolysis in cells subjected to hypoxia or simulated I/R. (8). In this proposal, we will explore the hypothesis that BnipS functions as a redox sensor that is activated by increased oxidative stress during I/R, leading to mitochondrial dysfunction and subsequent cell death. This hypothesis will be explored with the following specific aims: 1. Investigate the role of BnipS as a mitochondrial sensor of oxidative stress 2. Define the molecular mechanism(s) by which BnipS mediates mitochondrial fragmentation 3. Elucidate the role of autophagy in BnipS-mediated cell death 4. Characterize the role of BnipS proteolysis in response to I/R Our long-term goal is to understand the pathways that contribute to I/R injury and the results from this proposal will provide new insights into the pathways of apoptosis and their regulation in the heart. Further understanding of how BnipS functions in the heart has the potential to identify new therapeutic targets to treat or prevent heart disease.